A complete understanding of the molecular events that account for the transcellular transport of glucose from plasma to the interstitial fluid of brain will include identification of the transporters, their sites of localization, and the mechanisms that regulate their activities. In this competing continuation proposal, the applicant outlines such an investigation of the brain's glucose transporters at the mRNA and protein levels. He will synthesize 12-25 amino acid polypeptides corresponding to the known sequence of the C-terminal and of GLUT3 (a transporter isoform found in brain) and to the deduced sequences of any previous unknown transporters (identified by PCR amplification of a 35-base consensus transporter cDNA sequence derived from a canine brain mRNA preparation). These synthesized polypeptides and identified cDNAs will be used to prepare polyclonal antibodies and labeled oligonucleotides for light and electron microscopic immunocytochemistry/ELISA and in situ hybridization/Northern blots, respectively These reagents will allow (a) determination of the localization, abundance, and subcellular distribution of transporters in hippocampal endothelial, neuronal, and glial cells; and (b) study of the regulation of the transporters by ischemia in the bilateral carotid arterial occlusion model (gerbil) and by hypoglycemia in the insulin osmotic pump model (rat). Finally, the induction of GLUT1 (a transporter isoform found in brain endothelial cells) will be evaluated in vitro by subjecting cultured canine brain endothelial cells to a combination of extracellular matrix proteins and/or astrocyte-conditioned Matrigel. Active extracts will be fractionated by size and chromatographic properties and characterized. It is hoped that research describing the molecular events of blood-brain glucose transport will be valuable in the understanding of a wide range of neurological disorders.